Many signals are perceived by cells via ligand-receptor interactions at the interface between their plasma membranes and their micro-environment. Such communications relay diverse cues about development, nutrient availability or the presence of pathogens. Their importance is highlighted by the fact that membrane receptors are major drug targets. The activation of receptors by their cognate ligands is based on their reversible interaction, thereby forming a transient complex. The study of this complex is at the core of important research fields, including drug discovery. Classical methods for the study of protein-protein or ligand-receptor are generally laborious and often limited by the large amount of biological material required for analysis (X-ray diffraction, crystallography, mass spectrometry), the limited mass range (NMR-spectroscopy), the low-specificity (ultracentrifugation) or the low mass resolution (gel electrophoresis). An additional intrinsic problem in the characterization of non-covalently bound assemblies is that their analysis generally leads to the disruption of the weak and transient interactions. Hence, alternative or improved approaches that combine speed, accuracy and sensitivity for the detection of ligand-receptor interactions are actively sought after.
Chemical cross-linking results in the formation of artificial covalent bonds between the close interacting partners. It is a powerful method because it locks together receptors and their ligands normally associated only by weak and transient interactions, thereby enabling a wide range of analytical techniques that disrupt non-covalent bonds. Indeed, cross-linking techniques are nowadays extensively used for many proteomics methods including the analysis of protein structure and interactions, and for therapeutic use. Identifying unknown binding partners and specific interaction sites are among the most intense research.
Among the oldest cross-linking reagents are formaldehyde and glutaraldehyde. Due to their small size, they can penetrate through cell walls where they form cross-links between both proteins and nucleic acids, making them still one of the most efficient cross-linkers. However, glutaraldehyde and formaldehyde based cross-linking is not specific, resulting in many different complex products and modifications of the starting material. As a result, the cross-linked adducts are difficult to analyze, making the biological relevance of this cross-linking technique questionable.
More modern cross-link techniques take advantage of functional groups of which the intrinsic reactivity is only triggered by an external signal, such as light-irradiation. However, these functional groups are normally very bulky, disrupting normal protein-protein interactions or cannot be easily incorporated into a protein. Moreover, photo-activation cross-link techniques result on average in low cross-link yields and damage of the peptides under study. The smaller photo-reactive groups such as phenylazide, phenyldiazirine and benzophenone have major drawbacks. For instance, the phenylazide probes produce various by-products; the phenyldiazirine is only accessible through extensive and costly synthesis; and the benzophenone probe requires prolonged UV-irradiation, damaging and non-specifically cross-linking the starting material.
Although furan is commercially available, its use has always been limited in view of its toxicity and carcinogenicity. In the liver, cytochrome P450 catalyzes oxidation of furan to a reactive aldehyde, which subsequently reacts with sulfhydryl and amine groups.
WO 2010/068278 relates to the production of carrier-peptide conjugates through reaction between two chemically reactive unnatural amino acids. Thus, in this highly artificial system both binding partners must have unnatural amino acids, opposing the study of any natural protein-protein interaction.
Stevens and Madder describe furan-modified oligonucleotides for fast high-yielding and site selective DNA inter-strand cross-linking with non-modified complements (Stevens and Madder, Nucleic Acids Research, 2009, vol. 37(5), 1555-1565). Although the distances within the major and minor groove in the DNA duplex are known and fixed, and although the complementary (binding) nucleotides are known, Stevens and Madder describe a strong selectivity for cross-linking to either complementary A or C. In contrast, proteins are exceedingly more complex in each dimension, e.g. by the number of building blocks, conformations, cis-interactions, trans-interactions, transient interactions, anonymity of the binding partner, etc. Obviously, Stevens and Madder are completely silent on cross-linking of proteins.
Deceuninck et al. describe a strategy for peptide labeling on a solid support, relying on the incorporation of a furan moiety (Deceuninck et al., Chem. Commun., 2009, 21(3), 340-342). However, already the first step of providing peptides comprising a furan-moiety is arduous. Indeed, only after linking the dye to the peptide, the labeled peptide was cleaved from the solid support on which the peptide was synthesized. Schulz et al. (Protein and Peptide Letters, 2004, 11, 601-606) underscore the difficulties in achieving substantially pure furan-peptides by Fmoc-based solid phase synthesis. Apparently, Deceuninck et al. is wholly silent on protein-protein interactions.
N-bromo-succinimide (NBS) can be used for selective oxidation of furan rings, e.g. in site selective DNA inter-strand cross-linking (see Stevens and Madder ibid.). However, the use of NBS for selectively oxidizing peptides comprising a furan-moiety is counter intuitive, considering the sensitivity of various amino acids to oxidize as well as the known use of NBS to degrade proteins.
Therefore, there remains a need in the art to provide improved methods for cross-linking peptides. In addition, there remains a need in the art to improve methods for providing free furan-peptides.